A variety of human diseases, in their active stages, are characterized by migration of large numbers of neutrophils (PMN) through tissues and across mucosal surfaces. For example, in inflammatory pulmonary diseases, such as bronchitis, cystic fibrosis and bronchiectasis, acute inflammation of the airway is characterized by infiltration of bronchial epithelium with neutrophils. In the urinary system, migration of PMN across tubular and transitional epithelium is associated with cystitis and pyelonephritis. In the gastrointestinal tract, active inflammatory disease characterized by migration of PMN across the epithelial lining is the hallmark of chronic and self-limited diseases such as ulcerative colitis, Crohn's disease and bacterial eriterocolitis. In these conditions, epithelial injury, disease activity and symptoms parallel PMN infiltration of the mucosa. (Hawker, et al., Gastroenterology, 79: 508, 1980; Weiland, et al., Am. Rev. Respir. Dis., 133: 218, 1986; Nusrat, et al., Gastroenterology, 113: 1489, 1997; Koyama, et al., Immunol., 147: 4293, 1991).
The emigration of circulating PMN from the blood stream to mucosal surfaces is an essential component of the acute inflammatory response and involves a complex and incompletely understood series of events. During this process, there are sequential, bi-directional interactions of PMN with endothelial cells, interstitial matrix and, under many conditions, epithelial cells. During the initial phases of recruitment, circulating PMN are activated by exposure to inflammatory mediators including complement fragments, cytokines such as IL-8 and tumor necrosis factor, and lipopolysaccharide leading to their microvascular sequestration and firm adhesion to the endothelium (Pober, et al., Transplantation, 50: 537, 1990; Luscinskas, et al., J. Immunol., 146: 1617, 1991; Smith, et al., J. Immunology, 72: 65, 1991). Sequestered PMN then migrate across the vascular endothelium, a process that involves sequential interactions between PMN integrins and endothelial cell adhesion molecules including ICAM and PECAM (Smith, et al., Semin. Hematol., 30:45, 1993; Smith, et al., J. Clin. Invest., 83: 2008, 1990; Springer, Nature, 346: 425, 1990; Muller, et al., J. Exp. Med., 178: 449, 1993). In the extravascular space, directed PMN migration depends on the presence of a chemotactic stimulus and interactions between extracellular matrix proteins and adhesion molecules. Chemotactic stimuli can be produced locally by activated cells or by external pathogens. For example, intestinal epithelial cells secrete the chemoattractant IL-8 in a basolaterally polarized manner as a response to many pathogens (McCormick, et al., J. Cell Biol., 131: 1599, 1995) which then serves to recruit PMN from the circulation to the sub-epithelial space. PMN migration across the epithelium is then directed towards bacterial-derived peptides such as fMLF and requires sequential adhesive and signaling interactions along the basolateral epithelial cell membrane (Parkos, Bioessays, 19: 865, 1997).
While many of the molecular steps involved in this complex process of transmigration are still undefined, it has been demonstrated that a transmembrane protein termed CD47 plays an important role in regulating PMN migration at the level of the endothelium, matrix and epithelium (Parkos, et al., J. Cell Biol., 132: 437, 1996; Cooper, et al., Proc Natl Acad Sci USA, 92: 3978, 1995). In particular, it has been shown that CD47 is a crucial component of the transepithelial migration response and that CD47-dependent events occur after β2 integrin-mediated neutrophil adhesion to the epithelium (Parkos, et al., J. Cell Biol., 132: 437, 1996). Recently it was reported that CD47 binds to another class of transmembrane Ig superfamily members called Signal Regulatory Proteins (SIRPs) (Jiang, et al., J. Biol. Chem., 274: 559, 1999; Seiffert, et al., Blood, 94: 3633, 1999). SIRP's have been implicated in both positive and negative signal transduction cascades in a variety of cell types. CD47 was first purified from human placenta in 1990 (Brown, et al., J. Cell Biol., 111: 2785, 1990), and the complete cDNA sequence was reported by Campbell (Campbell, et al., Cancer Res, 52: 5416, 1992) and Lindberg et al. (Lindberg, et al., J. Cell Biol, 123: 485, 1993) and is widely expressed in hematopoietic cells (e.g., erythrocytes, lymphocytes, platelets, monocytes and neutrophils) and other tissues (e.g., placenta, surface epithelia, liver and brain). The human CD47 gene has been mapped to chromosome 3, band q 13.1-q 13.2 and encodes a protein of 305 amino acids (isoform 2) with a predicted core polypeptide molecular weight of 35 kDa and four isoforms have been cloned and characterized (Campbell, et al., Cancer Res, 52: 5416, 1992; Lindberg, et al., J. Cell Biol., 123: 485, 1993; Reinhold, et al., Journal of Experimental Medicine, 185: 119-21, 1997). The primary sequence of CD47 predicts an N terminal extracellular domain that structurally belongs to the immunoglobulin variable-like (IgV) superfamily (Lindberg, et al., J. Cell Biol., 123: 485, 1996; Vaughn, et al., Neuron 16(2): 261-73, 1996) containing a disulfide bridge harboring several potential N-glycosylation sites. Several hydrophobic helices suggest three or five transmembrane segments. There is a short hydrophilic intracytoplasmic tail with four alternatively spliced forms that are expressed in a tissue-specific manner (Campbell, et al., Cancer Res, 52: 5416, 1992; Lindberg, et al., J. Cell Biol., 123: 485, 1993; Reinhold, et al., Journal of Experimental Medicine, 185: 1, 1997). Studies with several IgV superfamily proteins have shown that the IgV-like domain is important in cell surface adhesive functions (Vaughn, et al., Neuron 16(2): 261-73, 1996). Studies using depletion and chimeric constructs suggest that the extracellular IgV-like domain of CD47 is important for its ability to regulate β3 integrin avidity for immobilized vitronectin (Lindberg, et al., J. Cell Biol., 123: 485, 1996). Direct association between CD47 and β3 integrin was first demonstrated by coimmunoprecipitation experiments in placenta (Brown, et al., J. Cell Biol., 111: 2785, 1990). These findings have further been validated by studies using CD47 knock out mice who rapidly die of Escherichia coli peritonitis, a phenomenon directly correlated with a reduction in leukocyte activation in response to β3 integrin ligation (Lindberg, et al., J. Cell Biol., 123: 485, 1996).
CD47 has been shown to have a central role in PMN transepithelial migration (Parkos, et al., J. Cell Biol., 132: 437, 1996). A potent CD47-specific antibody (Ab), C5/D5, was identified that was capable of inhibiting PMN migration across vascular endothelium, collagen-coated filters and intestinal epithelium without inhibiting β2 integrin-mediated adhesion (Parkos, et al., J. Cell Biol., 132: 437, 1996). At the same time, it was shown that anti-CD47 also inhibited PMN migration across endothelial monolayers (Cooper, et al., Proc Natl Acad Sci USA, 92: 3978, 1995). Subsequent studies with CD47 knockout mice have confirmed the importance of CD47 in PMN migration in vivo suggesting that CD47 plays a role in regulating the rate of PMN recruitment to sites of infection. (Lindberg, et al., Science, 274: 795, 1996)
Despite a growing number of reports of different functions of CD47 (Parkos, et al., J. Cell Biol., 132: 437, 1996; Jiang, et al., J. Cell Biol., 274: 559, 1999; Lindberg, et al., J. Cell Biol., 134: 1313, 1996; Ticchioni, et al., Journal of Immunology, 158: 677, 1997; Waclavicek, et al., Journal of Immunology, 159: 5345, 1997; Gao, et al., J. Cell Biol., 135: 533, 1996; Furusawa, et al., J. Biol. Chem., 123: 101, 1998; Frazier, et al., J. Biol. Chem., 274: 8554, 1999; Chung, et al., J. Biol. Chem., 272: 14740, 1997; Chung, et al., Blood, 94: 642, 1999), the mechanism by which CD47 regulates PAN migration is not known. In other cell systems, CD47 has been shown to functionally and physically associate with β3 and β1 integrins (Brown, et al., J. Cell Biol., 111: 2785, 1990; Lindberg, et al., J. Cell Biol., 134:1313, 1996; Frazier, et al., J. Biol. Chem., 274: 8554, 1999; Blystone, et al., J. Cell Biol., 130: 745, 1995; Lindberg, et al., J. Cell Biol., 123: 485, 1996). However, others have not been able to detect a direct association of CD47 with β1, β2, and β3 integrins in PMN and observe no significant effect on PMN transepithelial migration in the presence of a panel of functionally inhibitory mAbs against β3 integrin or β1 integrin (Liu, et al., J. Biol. Chem., 276: 40156). CD47 has also been shown to bind to the C-terminal cell binding domain of thrombospondin1 (Gao, et al., J. Biol. Chem., 271: 21, 1996), but this interaction does not modulate PMN migration (Liu, et al., J. Biol. Chem., 276: 40156, 2001). Recent in vitro studies have suggested that CD47 regulates the rate of PMN migration (Liu, et al., J. Biol. Chem., 276: 40156, 2001). In that study it was found that anti-CD47 mAbs delayed PMN migration across both T84 epithelial monolayers and matrix-coated permeable filters towards the chemoattractant fMLF. However, despite delayed transmigration, the numbers of PMN migrating across were not affected by the presence of anti-CD47 antibodies. This finding is consistent with studies with CD47 knock-out mice (Lindberg, et al., Science, 274: 795, 1996) suggesting that, although CD47 deficient PMN can eventually migrate to sites of infection, the delayed response resulted in enhanced mortality.
As indicated above, surface Ig superfamily member SIRPα (also termed SIRPα1; P84, Bit, SHPS-1 and MFR) (Jiang, et al., J. Biol. Chem., 274: 559, 1999; Seiffert, et al., Blood, 94: 3633, 1999; Vernon-Wilson, et al., European Journal of Immunology, 30: 2130, 2000; Kharitonenkov, et al., Nature, 386: 181, 1997; Han, et al., J. Biol. Chem., 275: 3798, 2000; Brumell, et al., J. Biol. Chem., 272: 875, 1997) was reported to bind to CD47. SIRPs are a family of transmembrane glycoproteins expressed in a variety of tissues (Kharitonenkov, et al., Nature, 386: 181, 1997). However, within these tissues, SIRPs are only selectively expressed in certain cell types (Adams, et al., Journal of Immunology, 161: 1853, 1998). In mice, SIRPs (termed SHPS-1) are richly expressed in hematopoietic cells including macrophages and myeloid cells, but not in T and B cells (Veillette, et al., J. Biol. Chem., 273: 22719, 1998). In humans, SIRPs are expressed in monocytes, granulocytes, dendritic cells and CD34+CD38−CD133+bone marrow stem/progenitor cells but not in lymphocytes (Seiffert, et al., Blood, 94: 3633, 1999; Seiffert, et al., Blood, 97: 2741, 2001). Through cDNA library screening, multiple homologous sequences that account for at least 15 additional SIRP members have been reported (Kharitonenkov, et al., Nature, 386: 181, 1997).
Primary structural analysis indicates that SIRPs all share common structural motifs that comprise a single transmembrane segment and an N-terminal extracellular domain that contains three Ig-like loops connected by three pairs of disulfide bonds. Therefore, SIRPs structurally belong to the Ig superfamily. The C-terminal intracellular domain, on the other hand, structurally separates two subfamilies of SIRPs termed SIRPα and SIRPβ (Kharitonenkov, et al., Nature, 386: 181, 1997). SIRPα has a long intracellular domain containing four tyrosine residues that form two immunoreceptor tyrosine-based inhibitory motifs (ITIM), while SIRPβ contains a basic lysine residue followed by a short intracellular tail that serves as a receptor for DAP12, a protein with an immunoreceptor tyrosine-based activation motif (Kharitonenkov, et al., Nature, 386: 181, 1997; Seiffert, et al., Blood, 97: 2741, 2001).
Similar to other ITIM domain containing proteins such as CD3, TCRξ, FcRγ, and BCR (Tomasello, et al., Seminars in Immunology, 12: 139, 2000; Vivier, et al., Immunology Today, 18: 286, 1997), SIRPα has been shown to play an important role in regulating cellular responses to a wide variety of different stimuli. For example, treatment of tissue-cultured cells with growth factors (including PDGF and EGF), growth hormone, insulin, CSF, LPA, etc., has been shown to induce phosphorylation of tyrosines on the intracellular ITINI domain of SIRPα resulting in binding to SH2 domain containing tyrosine phosphatase-1 or 2 (SHP-1 and 2) (Kharitonenkov, et al., Nature, 386:181, 1997; Veillette, et al., J. Biol. Chem., 273: 22719, 1998; Timms, et al., Molecular & Cellular Biology, 18: 3838, 1998). SIRPα binding to SHP-1 or SHP-2 has been shown to deliver positive or negative signals that regulate a variety of cellular functions, respectively (Kharitonenkov, et al., Nature, 386:181, 1997; Stofega, et al., J. Biol. Chem., 275: 28222, 2000; Stofega, et al., J. Biol. Chem., 273: 7112, 1998).
As mentioned above, there are recent reports of SIRPα1 as an extracellular ligand for CD47. These findings are important in that no membrane protein receptor has been described for either SIRP or CD47. CD47-SIRPα interactions have been implicated in a number of cellular functions, including the regulation of neutrophil migration, memory formation, macrophage multinucleation, B cell aggregation, T cell activation, dendritic cell maturation and function, and red blood cell self-recognition in mice, (Kharitonenkov, et al., Nature, 386: 181, 1997; Han, et al., J. Biol. Chem., 275: 37984, 2000; Chang, et al., Learning & Memory, 6: 448, 1999; Oldenborg, et al., Science, 288: 2051, 2000; Tanaka, et al., Journal of Immunology, 167(5): 2547-54, 2001; Liu, et al., v 277(12): 10028-36, 2002; Avice, et. al., Journal of Immunology, 167(5): 2459-68, 2001; Blazar, et al., Journal of Experimental Medicine, 194(4):541-9, 2001; Seiffert, et al., Blood, 97(9): 2741-9, 2001; Oldenborg, et al., Journal of Experimental Medicine, 193(7): 855-62, 2001; Pettersen, Apoptosis, 5(4): 299-306, 2000; Han, et al., J. Biol. Chem., 275(48): 37984-92, 2000; Demeure, et al., Journal of Immunology, 164(4): 2193-9, 2000; Armant, et al., Journal of Experimental Medicine, 190(8): 1175-82, 1999; Jiang, et al., J. Biol. Chem., 274(2): 559-62, 1999; Lee, et al., European Journal of Neuroscience, 12(3): 1105-12, 2000; and Chang, et al., Learning & Memory, 6(5): 448-57, 1999) although the mechanisms remain undefined.
Thus, a heretofore unaddressed need exists in the industry to modulate the interaction between CD47 and SIRPα.